Methods of detecting sequences of nucleic acids are of wide applicability in research and medical fields. Nucleic acid analysis has been applied in areas such as detection of single nucleotide polymorphisms (SNP's), infectious disease screening, diagnosis and prognosis of genetic disease and assessment of treatment. The ability to identify nucleic acid sequences at the single base level for an increasing number of positions within a particular genome is required.
The demand for nucleic acid-based technologies with diagnostic and microarray capabilities is on the rise. This can be partly attributed to the Human Genome Project, which has been instrumental in prompting further investigation into such areas as the genetic basis of disease, genetic predisposition to disease and pharmacogenomics. There is a need for easy-to-use, low-cost, clinically relevant tests that are highly sensitive and specific.
Hybridization, the intermolecular association of nucleic acid molecules through hydrogen bonding of nucleic acid bases between molecules underlies many of the most promising analytical techniques. The overall success of any hybridization-based assay relies on a number of factors. In an ideal system, the hybridization is very sensitive, i.e., hybridization between a capture moiety and its target occurs readily. The hybridization should also be very specific, i.e., hybridization between the capture moiety and molecules which are not a target can be largely avoided.
Molecular biological techniques have been developed which employ enzyme-mediated target amplification strategies to increase the copy number of a specific analyte. This generally increases the ease with which the amplified analyte can be detected or otherwise manipulated. Examples of such techniques include the polymerase chain reaction (PCR), ligase chain reaction (LCR), transcription-mediated amplification (TMA), strand-displacement amplification (SDA) and nucleic-acid sequence-based amplification (NASBA). However, all of these technologies are based on linear probe sequences, and have their limitations particularly with respect to issues relating to specificity. This is because incorrect hybridization can lead to amplification of an undesired analyte.
Attempts have been made, with varying degrees of success, to increase the sensitivity and specificity of nucleic acid hybridization processes. For example, Lane et al. have suggested a nucleic acid capture moiety that includes a hairpin duplex adjacent to a single stranded region complementary to a target sequence. Further, the inclusion of an element capable of stabilizing the intermolecular duplex, once formed was suggested (NAR 1997; 25: 611–16, U.S. Pat. No. 5,770,365). Other approaches include nucleotide analogues that enhance thermal stability differences with the idea of improving the discrimination of single nucleotide polymorphisms. Adjusting buffer components, temperature; electrical potential etc. have also been used to enhance mismatch discrimination.
Current technologies which control for specificity of hybridization rely mainly on modification of environmental conditions such as temperature, salt concentration, addition of DNA-specific condensing (TMAC) or denaturing agents (formamide). These technologies, while adequately controlling individual nucleic acid tests, lack the ability to control complex mixtures of DNA tests to the same level of accuracy. For instance, temperature for hybridization needs to be controlled closely, preferably to better than +/−1° C. However, differences in base composition of probe moieties continue to make it difficult to obtain optimum conditions for the use of many probes in a single mixture. To reduce Tm differences associated with nucleotide compositions of probe moieties, chaotropic agents have been used. Quaternary or tertiary amine salts such as tetramethylammoniumchloride (TMAC) have been used with some success.
Duplex denaturing reagents, such as formamide, can increase the specificity of target binding to its cognate probe capture moiety. In this approach, a duplex denaturant is used to destabilize duplex formation, particularly duplexes resulting from hybridization of mismatched nucleic acid sequences.
Also attempted, has been the design of sequences which minimally cross-hybridize with each other. Such families of sequences can be used as ‘zipcodes’, ‘barcodes’ or ‘tags’ that are associated with the target and subsequently hybridized to the anti-tag (tag complement) found on the microarray, bead, etc. Families of nucleic acid tags wherein each member of the tag family varies from every other member of the family by a particular minimum number of bases (comparing tags in end-to-end alignment) have been described. See, for example, U.S. Pat. No. 5,604,097, Brenner, U.S. Pat. No. 5,635,400, Brenner; U.S. Pat. No. 5,654,413, Brenner, U.S. Pat. No. 6,138,077, Brenner; U.S. Pat. No. 6,150,516, Brenner et al.; U.S. Pat. No. 6,172,214, Brenner; and U.S. Pat. No. 6,235,475, Brenner et al.
Another probe, called a molecular beacon, is a single stranded stem and loop structure with a fluorophore attached to one end and a quencher attached to the other (Tyagi et al. U.S. Pat. No. 5,925,517). The principle for target detection is based on the hybridization of the target sequence to the single stranded loop forcing the stem to unwind resulting in fluoresence. Optimal position of the mismatch within the loop region has been studied (Bonnet et al. Proc. Natl. Acad. Sci. USA. 1999. 96:6171–6176).
Especially valuable to modem genomics technologies, such as biochips or DNA microarrays that process larger numbers would be probes that permit many (100's to 100,000's) of tests to be run in parallel. Also valuable would be probes that reliably discriminate between sequences that differ from each other by only one nucleotide, such as SNPs.